The invention relates to the use of Toso proteins in screening assays.
Apoptosis or programmed cell death is an important homeostatic mechanism that maintains cell number, positioning, and differentiation. Several intracellular and intercellular processes are known to regulate apoptosis. One of the best characterized systems is initiated by the cell surface receptor, Fas (Apo-1/CD95), homologues of which initiate apoptosis in a wide range of organisms (Itoh, et al., Cell, 66:233-243 (1991); Yonehara, et al., J. Exp. Med., 169:1747-1756 (1989)). Clustering of the Fas cytoplasmic domain generates an apoptotic signal via the xe2x80x9cdeath domainxe2x80x9d (Itoh and Nagata, J. Biol. Chem., 268:10932-10937 (1993)). Several critical proteins that bind to the death domain or other domains within the cytoplasmic region have been identified using yeast two-hybrid and biochemical screens (Boldin, et al., J. Biol. Chem., 270:7795-7798 (1995); Chinnaiyan, et al., Cell, 8145:505-512 (1995); Chu, et al., Proc. Natl. Acad. Sci. USA, 92:11894-11898 (1995); Okura, et al., J. Immunol., 157:4277-4281 (1996); Sato, et al., Science, 268:411-415 (1995); Stanger, et al., Cell, 8145:513-523 (1995)).
Fas engagement by Fas ligand is capable of activating the interleukin-1 xcex2 converting enzyme family of cysteine proteases (Caspases)xe2x80x94the proteolytic executors of apoptosis (Enari, et al., Nature, 375:78-81 (1995); Enari, et al., Nature, 380:723-726 (1996); Los, et al., Nature, 375:81-83 (1995); Tewari and Dixit, J. Biol. Chem, 270:3255-3260 (1995)). Recent studies implicate caspase8 (MACH/FLICE/Mch5) as linking Fas receptor signaling to downstream caspases via its association with FADD/MORT1 (Boldin, et al., (1995); Chinnaiyan, et al., (1995); Boldin, et al., (1996); Fernandes-Alnemri, et al., Proc. Natl. Acad Sci. USA, 93:7464-7469 (1996); Muzio, et al., Cell, 85:817-827 (1996)). Several groups have reported that caspase-8 activation is inhibited by a cellular inhibitor, cFLIP/FLAME-1/1-FLICE (Irmler, et al., Nature, 388:190-195 (1997); Srinivasula, et al., J. Biol. Chem., 272:18542-18545 (1997); Hu, et al., J. Biol. Chem., 272:17255-17257 (1997)). Other proteins involved in Fas-mediated apoptosis include: (a) the Fas-activated serine/threonine kinase (FAST kinase), which is rapidly activated during Fas-mediated apoptosis; (b) acid sphingomyelinase, which produces ceramide, a pro-apoptotic signal that acts as a second messenger in several systems; and (c) Daxx, a novel protein that links Fas to the JNK stress kinase pathway (Cifone, et al., J. Exp. Med., 180:1547-1552 (1994); Tian, et al., J. Exp. Med., 182:865-874 (1995); Yang, et al., Cell, 89:1067-1076 (1997)). The exact role of these latter co-activators has yet to be fully defined.
A balance between life and programmed cell death signals in cells is likely to be governed by multiple interacting regulators that counteract apoptotic signals with appropriate anti-apoptotic signals. Imbalances in this regulation can result in wide variety of pathologies, including cancer and immune dysfunction and it is now clear that other polypeptides besides Fas contribute to disregulation of appropriately induced apoptosis. As an example, in many tumor cell lines Fas expression does not correlate with sensitivity to Fas-induced apoptosis, implying the existence of Fas-resistance protein (Richardson, et al., Eur. J. Immunol., 24:2640-2645 (1994)). Also, in some types of cells, Fas-induced apoptosis requires protein synthesis inhibitors such as cycloheximide (Itoh and Nagata, (1993); Yonehara, et al., (1989)) and even in Fas-sensitive cells, protein synthesis inhibitors can play a synergistic role with cycloheximide (Itoh and Nagata, (1993)). These combined observations further suggest the existence of proteins capable of suppressing Fas-generated apoptotic signaling.
Additionally, in the course of a normal immune response, both cytotoxic T cell and NK cell activation can lead to Fas ligand (FasL) induction of apoptosis in target cells (Arase, et al., J. Exp. Med., 181:1235-1238 (1995); Berke, Cell, 81:9-12 (1995); Montel, et al., Cell Immunol., 166:236-246 (1995)). Although both Fas and FasL are rapidly induced following T-cell activation, activated-T cells remain resistant to Fas-induced apoptosis for several days (Klas, et al., Int. Immunol., 5:625-630 (1993); Owen-Schaub, et al, Cell Immunol., 140:197-205 (1992)). Thus, a mechanism exists to shield newly activated T cells from the cytotoxicity of their own FasL expression. This is likely to be an important component of T cell activation processes and protection in lymph nodes, splenic germinal centers and other sites at which T cell activation results in apoptosis of target cells.
Described herein is the identification and characterization of a novel surface molecule, xe2x80x9cTosoxe2x80x9d which is a member of the immunoglobulin gene superfamily and which specifically inhibits Fas and TNF receptor family mediated apoptosis. The results demonstrate the existence of cell surface mediated signaling pathways that lead to down regulation of Fas-mediated apoptosis in certain cell types and suggest that activation of T cells suppresses internal signaling systems that might lead inappropriately to T cell-induced self-killing.
Accordingly, it is an object of the invention to provide Toso proteins and related molecules. It is a further object of the invention to provide recombinant nucleic acids encoding Toso proteins, and expression vectors and host cells containing the nucleic acid encoding the Toso protein. A further object of the invention is to provide methods for screening for antagonists and agonists of Toso.
In accordance with the objects outlined above, the present invention provides methods for screening for a bioactive agent capable of binding to a Toso protein encoded by a recombinant nucleic acid that will hybridize under high stringency conditions to the nucleic acid sequence depicted in FIG. 1 (SEQ ID NO:1) or its complement. The methods comprise combining a Toso protein and a candidate bioactive agent, and determining the binding of the candidate agent to the Toso protein.
In an additional aspect, the invention provides methods for screening for a bioactive agent capable of modulating the activity of a Toso cell-surface receptor, said method comprising the steps of adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a Toso receptor, exposing the cells to an apoptotic agent that will induce apoptosis, and determining the effect of the candidate bioactive agent on apoptosis.
In a further aspect, the invention provides methods of modulating apoptosis in a cell comprising administering to the cell an exogenous compound that binds to a Toso protein wherein the binding modulates the biological activity of said Toso protein.
In an additional aspect, the invention provides methods for identifying a cell containing a mutant Toso gene comprising determining the sequence of all or part of at least one of the endogenous Toso genes. Similarly, methods of identifying the Toso genotype of an individual are provided.
In a further aspect, the invention provides methods for diagnosing an apoptosis related condition in an individual. The activity of Toso in a tissue from a first individual is measured and compared to the activity of Toso in a tissue from a second, unaffected individual or from a second tissue in the first individual. When the activity of Toso from said first individual is less than the activity of Toso in the second individual, the first individual is at risk for an apoptosis related condition.